Test system for evaluating the compatibility of biologically active substances with copolymers

ABSTRACT

A test solution agent, a test system and a method for evaluating the compatibility of biologically active substances with N-vinylpyrdolidone are disclosed.

The present invention relates to a liquid mixture serving as testsolvent, and to a test system and a method for evaluating thecompatibility of biologically active substances with N-vinylpyrrolidonecopolymers using the liquid mixture.

Solid dispersions, i.e. homogeneous microdisperse phases of two or moresolids and the special case of so-called solid solutions (moleculardispersion systems), and their use in pharmaceutical technology aregenerally known, cf. Chiou and Riegelman J. Pharm. Sci., 60, 1281-1300(1997).

Solid solutions can be produced by melting processes or by the solutionprocess. Particularly suitable as polymeric excipient for producing suchsolid dispersions or solid solutions are N-vinylpyrrolidone copolymers,i.e. copolymers of N-vinylpyrrolidone with further ethylenicallyunsaturated monomers. Solid solutions of biologically active substancesbased on such copolymers can be produced particularly advantageously bymelt extrusion as described, for example, in EP-A 240 904.

However, there are minimum requirements on the amounts employed toproduce melt extrudates. If only relatively small amounts of activeingredient are available, it cannot be predicted with certainty whetheran active ingredient will form a solid solution together with the chosencopolymer. However, it is precisely when drug products based on newactive ingredients are being developed that only relatively smallamounts of the active ingredient are frequently available, so that thepossibility of prediction with the aid of a simple test system appearsto be extremely desirable.

It is likewise desirable to be able to make predictions concerning thestability of solid solutions or solid dispersions. This is because,depending on the compatibility of the active ingredient with thecopolymer, the previously homogeneous disperse phase may becomeinhomogeneous, or the active ingredient may recrystallize. Such phaseseparation or recrystallization is unwanted because of the change in thehomogeneity and the release characteristics associated therewith.

EP-A 0987549 discloses a test system for characterizing thecompatibility of biologically active substances withpolyvinylpyrrolidone in a solid dispersion.

It is the object of the present invention to indicate a test system withthe aid of which it is possible to predict the compatibility ofbiologically active substances and N-vinylpyrrolidone copolymers in asimple manner.

It has surprisingly been found that the dissolving properties ofN-vinylpyrrolidone copolymers can be simulated by a liquid mixture of1,3-bis(pyrrolidon-1-yl)butane with certain compounds which havestructural similarity to the comonomer units present in the copolymer.

The invention therefore relates to a liquid mixture which comprises

-   a) 1,3-bis(pyrrolidon-1-yl)butane and-   b) at least one compound of the formula I-    in which-   Q is CH₂-Z, CH₂—CH₂-Z or CHZ-C₁-C₄-alkyl,-   n is 0, 1, 2 or 3, and-   the Z radicals are C₁-C₂o-alkylcarbonyloxy, carboxyl,    C₁-C₂₀-alkyloxycarbonyl, C₂-C₄-hydroxyalkyloxycarbonyl,    di(C₁-C₄-alkyl)amino-C₂-C₄-alkyloxycarbonyl or tri(C₁-C₄-alkyl)    ammonium-C₂-C₄-alkyloxycarbonyl.

All the Z radicals occurring in formula I are preferably identical.“Liquid mixture” is intended for the purposes of the present applicationto mean that the mixture is in liquid form at least at slightly elevatedtemperature, e.g. at 45° C., preferably even at room temperature.

The liquid mixture serves as test solvent which simulates thedissolving, properties of the N-vinyl-pyrrolidone copolymer. The liquidmixture normally contains components a) and b) in a ratio of from 10:1to 1:10, preferably 5:1 to 1:5, by weight.

The invention additionally relates to a test system for evaluating thecompatibility of a biologically active substance with a copolymer whichincludes units of N-vinylpyrrolidone and of at least one ethylenicunsaturated monomer of the formula IICH₂═CR-Z′  (II)in which R is hydrogen or methyl, and Z′ has the meaning indicated abovefor Z, where the test system includes the liquid mixture defined aboveand at least one biologically active substance.

The invention additionally relates to a method for evaluating thecompatibility of a biologically active substance with anN-vinylpyrrolidone copolymer, where the copolymer includes units ofN-vinylpyrrolidone in a proportion by weight of xvp and units of atleast one ethylenically unsaturated monomer of the formula IICH₂═CR-Z′  (II)in which R is hydrogen or methyl, Z′ is C₁-C₂₀-alkyl-carbonyloxy,carboxyl, C₁-C₂₀-alkyloxycarbonyl, C₂-C₄-hydroxyalkyloxycarbonyl,di(C₁-C₄-alkyl)amino-C₂-C₄-alkyloxycarbonyl or tri(C₁-C₄-alkyl)ammonium-C₂-C₄-alkyloxycarbonyl, in a proportion by weight of x_(M), inwhich

-   a) a test solvent which comprises 1,3-bis(pyrrolidon-1-yl)butane in    a proportion by weight of x_(VP) and a compound of the formula I in    a proportion by weight of x_(M)-    in which Q is CH₂-Z, CH₂—CH₂-Z or CHZ-C₁-C₄-alkyl, n is 0, 1, 2 or    3, the Z radicals are identical and correspond to the Z′ radical, is    prepared,-   b) the biologically active substance is brought into contact with    the test solvent, and-   c) the phase behavior of the mixture and/or solubility of the    biologically active substance in the test solvent is determined.

X_(VP) is in general from 10 to 90% by weight, usually 30 to 70% byweight. x_(M) is in general from 90 to 10% by weight, usually 70 to 30%by weight. If more than one monomer of the formula II is present, theindividual contribution of the various monomers x_(M1), x_(M2), . . .are to be put for x_(M).

The radicals Z and Z′ are preferably C₁-C₄-alkyl-carbonyloxy, carboxyl,C₁-C₄-alkyloxycarbonyl or C₂-C₄-hydroxyalkyloxycarbonyl. If Z or Z′ aretri(C₁-C₄-alkyl) ammonium-C₂-C₄-alkyloxycarbonyl, they are accompaniedby one equivalent of a pharmaceutically acceptable anion such ashydroxide, sulfate, hydrogen-sulfate, carbonate, hydrogencarbonate, ahalide, in particular chloride, the anion of an organic acid such asacetate, lactate, fumarate, or the like. If Z or Z′ are carboxyl, thecarboxyl group may also be wholly or partly neutralized, in which casesuitable charge-balancing cations are pharmaceutically acceptablecations such as alkali metal or alkaline earth metal ions, e.g. sodiumor potassium, or unsubstituted or substituted ammonium ions such asdimethylammonium, trimethylammonium, diethanolammonium and the like.

1,3-Bis(pyrrolidon-1-yl)butane can be obtained by dimerization ofN-vinylpyrrolidone under acidic reaction conditions and subsequenthydrogenation of the resulting 1,3-bis(pyrrolidon-1-yl)butene to1,3-bis-(pyrrolidon-1-yl)butane (cf. Breitenbach et al.,Naturwissenschaften 42, 955, 155; 440). 1,3-Bis-(pyrrolidon-1-yl)butaneis a colorless oily liquid with a boiling point of 205 to 215° C. (0.2mbar).

The compounds of the general formula I are either commercially availableor can be prepared in a simple manner. Use for example of1,3-diacetoxybutane and, in particular, 1,4-diacetoxybutane as compoundof the formula I has been successful. Compounds of the formula I can beobtained for example by esterification of polyols such as1,3-butanediol, 1,4-butanediol or 1,3,5-pentanetriol with carboxylicacids such as acetic acid or derivatives thereof or by esterification ofpolycarboxylic acids such as glutaric acid or adipic acid with suitablealcohols.

Suitable copolymers whose compatibility with biologically activesubstances can be evaluated with the aid of the test system of theinvention are copolymers of N-vinylpyrrolidone with ethylenicallyunsaturated monomers of the formula IICH₂═CR-Z′  (II)in which R is hydrogen or methyl, and Z′ is C₁-C₂₀-alkylcarbonyloxy,carboxyl, C₁-C₂₀-alkyloxycarbonyl, C₂-C₄-hydroxyalkyloxycarbonyl,di(C₁-C₄-alkyl)amino-C₂-C₄-alkyloxycarbonyl ortri(C₁-C₄-alkyl)ammonium-C₂-C₄-alkyloxycarbonyl.

Monomers of the formula II which may be mentioned are vinyl esters ofC₁-C₂₀-alkanecarboxylic acids, such as vinyl acetate or vinylpropionate, acrylic or methacrylic acid, C₁-C₂₀-alkyl esters of acrylicacid or methacrylic acid, such as methyl acrylate, methyl methacrylate,ethyl acrylate, ethyl methacrylate, C₂-C₄-hydroxyalkyl(meth)acrylatessuch as hydroxyethyl acrylate,di(C₁-C₄-alkyl)amino-C₂-C₄-alkyl(meth)-acrylates such asdimethylaminopropyl acrylate, or(meth)acryloyloxy-C₂-C₄-alkyltri(C₁-C₄-alkyl)ammonium salts such asacryloyloxypropyltrimethylammonium chloride.

The preferred copolymers include those of N-vinyl-pyrrolidone and vinylacetate, especially in a ratio of 70:30 to 30:70 by weight; andcopolymers of N-vinyl-pyrrolidone with methyl methacrylate, especiallyin a ratio of from 20:80 to 55:45 by weight.

The copolymers generally have a Fikentscher K value of from 10 to 110,in particular from 20 to 80.

The radical Z in the compound of the formula I is chosen in accordancewith the radical Z′ in the comonomer of the copolymer to be simulated.Thus, for example, a mixture of 1,3-bis(pyrrolidon-1-yl)butane with1,4-diacetoxybutane serves to simulate the dissolving properties ofN-vinylpyrrolidone/vinyl acetate copolymers. It is, of course, possiblefor the copolymer to be simulated also to contain two or more differentmonomers of the formula II. The test solvent is then prepared by usingtwo or more different compounds of the formula I with appropriatelychosen radicals Z as component b).

Compatibility means for the purposes of the present application theability of a substance to form with the N-vinylpyrrolidone copolymer ahomogeneous, stable solid dispersion, this solid dispersion being inparticular a solid solution, i.e. a molecular dispersion of thecomponents in one another. The test system is suitable in principle forall active pharmaceutical ingredients, crop protection agents, foodsupplements or cosmetic active ingredients. It is also possible toinvestigate detergents or dyes for their compatibility with thecopolymers. The influence of formulation aids which are not themselvesbiologically active, such as sugars, sugar alcohols, solubilizers suchas surfactants, or other polymeric aids, can also be investigated.

The method of the invention is carried out by first preparing a testsolvent. The test solvent comprises 1,3-bis(pyrrolidon-1-yl)butane and acompound of the formula I in a ratio by weight which corresponds to thatof N-vinylpyrrolidone and comonomer(s) in the copolymer to be simulated.The solubility of the biologically active substance to be investigatedin the liquid mixture is then assessed at a defined temperature, usuallyroom temperature. The solubility can be determined quantitatively, e.g.in % by weight based on the weight of test solvent and biologicallyactive substance. In many cases, it is sufficient to state whether thesolubility is greater or less than a particular value. For this purpose,a predetermined amount of the biologically active substance is broughtinto contact with the test solvent. The quantitative ratios can inprinciple be chosen freely. However, it is advisable to choose theconcentration ranges in the test system such that they correspond to theactive ingredient content typical of extrudate forms, i.e. generallyfrom 0.1 to 70% by weight, preferably 10 to 30% by weight, ofbiologically active substance, based on the total weight of the testsystem.

The biologically active substance is normally weighed out, mixed withthe test solvent and preferably blended, e.g. stirred with a laboratorymagnetic stirrer at from 5 to 2000 rpm, or treated with ultrasound or avortex homogenizer. Dissolving can also be speeded up by heating thetest system. The heating preferably takes place in such a way that theheating rate approximately corresponds to that in a melt formulation,i.e. at from 0.5 to 5° C./min. The test system is preferably heated to amaximum of 140° C., e.g. to a temperature in the range from 45 to 140°C. or 110 to 140° C. However, heating to the boiling point of the liquidmixture is also possible in the individual case. The test system is thenallowed to cool to the determination temperature, usually roomtemperature.

The phase behavior of the mixture is then assessed, i.e. it isestablished by visual, spectroscopic and/or thermoanalyticalinvestigation of the resulting mixture whether the biologically activesubstance is able to form a homogeneous phase with the liquid mixture.

Visual inspection takes place for example using a microscope such as ausual optical microscope. It is established in this case whether a clearsolution has formed. Besides visual inspection, also suitable is aspectroscopic investigation of the test system. For example, the testsystem can be investigated for its amorphous character with the aid ofconfocal Raman spectroscopy. Also suitable is the method of differentialscanning calorimetry. It is possible to conclude from the presence of ahomogeneous phase that the solubility of the biologically activesubstance is greater than the concentration of the substance in thedissolving test. Conversely, a lower solubility can be concluded fromthe occurrence of a phase separation.

Quantitative determination of the solubility is possible for example inthe following way:

-   a) A concentration series is prepared by bringing various amounts of    biologically active substance into contact with a constant amount of    test solvent in parallel tests. After an equilibration time over a    defined period at a given temperature, preferably with blending,    e.g. stirring for 24 hours or ultrasound treatment for 30 min, the    maximum concentration at which clear solutions are obtained is    found. The solubility of the biologically active substance is    between the concentration at which a clear solution is obtained but    at the next concentration no clear solution is obtained.-   b) An amount of the test solvent which is insufficient for    completely dissolving is added to an amount of the biologically    active substance, or further amounts of biologically active    substance are added to the solution until the added amount no longer    completely dissolves. After an equilibration time over a defined    period at a given temperature, preferably with blending, e.g.    stirring for 24 hours or ultrasound treatment for 30 min, a sample    of the clear supernatant is taken. The mixture can for this purpose    be previously centrifuged, e.g. using an ultracentrifuge at 8000 to    12 000 rpm. The concentration of the biologically active substance    is determined in the sample of the clear supernatant, e.g. by high    pressure liquid chromatography (HPLC). The value which is found    corresponds to the solubility of the biologically active substance.

It is possible by means of the methods mentioned above to determine,depending on the equilibration conditions, the thermodynamic saturationsolubility or the maximum (kinetic) solubility.

The solubility found after 24 hours at room temperature (22° C.±2° C.)essentially corresponds to the thermo-dynamic saturation solubility ofthe biologically active substance. This value of the solubility is ameasure of the thermodynamic saturation solubility of the biologicallyactive substance in the matrix of the copolymer at room temperature.Solid solutions of biologically active substances are thermodynamicallystable if the active ingredient loading is below the thermodynamicsaturation solubility of the biologically active substance in thematrix.

However, the active ingredient loading in solid solutions can beincreased greatly by energy input. The maximum active ingredient loadingachievable in a given matrix of a copolymer can be predicted with thetest system of the invention by determining the solubility of thebiologically active substance in accordance with one of theaforementioned methods a) or b), the equilibration being carried out byheating to a temperature of, for example, 140° C., in particular from110 to 140° C., or by sonication.

The test system of the invention also allows the recrystallizationbehavior to be predicted. Especially for test systems in which higheractive ingredient loadings than the thermodynamic saturation solubilityhave been set up by energy input, e.g. heating or sonication, therecrystallization behavior after the energy input ceases or aftercooling to room temperature represents an important criterion. Testsystems in which the biologically active substance does notrecrystallize immediately are investigated for long-term stability. Itis possible to use the following conditions for this, for example:

-   -   leaving to stand at room temperature for 24 hours,    -   storage for one month, 3 months, 6 months in climate zone 2 (25°        C., 60% relative humidity) or climate zone 4 (20° C., 70%        relative humidity), or    -   stress storage at 40° C., 75% relative humidity for up to 6        months.

It is also possible with the aid of the test system of the invention toinvestigate the influence of excipients or a solubility-increasing orsolubility-reducing effect of the presence of a second or furtherbiologically active substance on the solubility of a first biologicallyactive substance in the matrix of the copolymer. For this purpose, theseexcipients, e.g. solubilizers, or further biologically active substancesare added to the test solvent in addition to the biologically activesubstance to be tested. It is then possible again to determine thethermodynamic saturation solubility and/or the maximum solubility asindicated above.

The invention is illustrated in more detail by the following examples.

EXAMPLE 1 Thermodynamic Saturation Solubility of Lopinavir in a Matrixof a Copolymer of N-vinyl-pyrrolidone and vinyl acetate (60:40)

The copolymer was simulated by a mixture of1,3-bis-(pyrrolidon-1-yl)butane and 1,4-diacetoxybutane in the ratio of6:4 by weight.

Method a)

In parallel tests, the active ingredient was weighed into glass bottlesand made up with the test solvent appropriate for the concentrationsindicated in the table below (all concentrations in weight/weight). Allseven samples were provided with a magnetic stirring bar and stirred atroom temperature for 24 hours. It was established by visual inspectionthat the active ingredient formed clear solutions up to 24.1%; the testwith 26.0% showed incomplete dissolving. The saturation solubility wastherefore between 24.1 and 26.0% by weight. Lopinavir 20.2 21.9 24.126.0 29.5 33.9 36.3 [%] Clear + + + − − − − solutionMethod b)

In an alternative determination method, 150 mg of lopinavir were mixedwith 350 mg of the test solvent and stirred at room temperature for 24hours. The sample was then centrifuged at 12 000 rpm for one minute. Theclear supernatant was investigated by HPLC. A solubility of 24.9% byweight was found.

Method c)

The maximum solubility was determined by mixing 60 mg of lopinavir with140 mg of the test solvent and treating with ultrasound at roomtemperature for 30 min. The solvent was then centrifuged at 10 000 rpmfor 10 min. A sample of the clear supernatant was investigated by HPLC.A maximum solubility of 39.72% by weight was found. The remaining amountof the sample was stored at room temperature for 4 weeks. Then a furthersample of the supernatant was taken and investigated by HPLC. Aconcentration of 28.87% by weight was found.

EXAMPLE 2 Thermodynamic Saturation Solubility of Lopinavir under theInfluence of polyoxyethylene-glycerol trihydroxystearate (Cremophor RH40®)

Example 1a) was repeated but with use of the concentrations indicated inthe table below, and with the test solvent containing 5% by weightCremophor RH 40. The saturation solubility was found to be between 22.0and 26.0%. Lopinavir 20.0 22.0 23.7 26.0 30.0 33.0 35.8 [%] Cremophor 55 5 5 5 5 5 [%]* Clear + + + − − − − solution*based on the test solvent

Example 1b) was repeated but with the test solvent containing 5% byweight Cremophor RH 40. A solubility of 22.8% was found.

It was possible to show with the aid of the test system of the inventionthat Cremophor RH 40 reduces the thermodynamic saturation solubility oflopinavir.

EXAMPLE 3 Thermodynamic Saturation Solubility of Lopinavir under theInfluence of Ritonavir

An active ingredient premix of the two abovementioned active ingredientsin a ratio of 4:1 by weight was prepared. Example 1a) was repeated withthis active ingredient mixture, the concentration being as in the tablebelow. Lopinavir [%] 19.2 20.6 23.9 27.8 Ritonavir [%]  4.8  5.2 6   6.9Total active 24.0 25.8 29.9 34.7 ingredient [%] Clear solution + + − −

Example 1b) was repeated with the abovementioned active ingredientpremix. 21.3% by weight lopinavir was found in the clear supernatant byHPLC. It was possible with the aid of the test system of the inventionto show that the presence of ritonavir reduces the thermodynamicsaturation solubility of lopinavir.

EXAMPLE 4 Thermodynamic Saturation Solubility of Lopinavir under theInfluence of Ritonavir and Cremophor RH 40

Example 3a) was repeated, using the concentrations indicated in thetable below, and with the test solvent containing 10% by weightCremophor RH 40. Visual determination of the concentration series led toa thermodynamic saturation solubility for the active ingredient mixtureof between 23.6% and 28.0%. The HPLC method led to a result of 21.7% forlopinavir. Lopinavir [%] 14.2 15.8 18.9 22.4 Ritonavir [%] 3.6 4.0 4.75.6 Total active 17.8 19.8 23.6 28.0 ingredient [%] Cremophor [%]* 10 1010 10 Clear solution + + + −*based on the test solvent

EXAMPLE 5 Melt Extrusion

A formulation containing 18.7 parts by weight of lopinavir, 4.7 parts byweight of ritonavir, 10.0 parts by weight of Cremophor RH 40 and 100parts by weight of N-vinylpyrrolidone/vinyl acetate copolymer (60:40)was prepared for the melt extrusion. Melt extrusion of this formulationusing a heated twin screw extruder resulted in stable solid solutions.After storage of the extrudates at room temperature for 8 months theyunderwent X-ray investigation. Both active ingredients were in X-rayamorphous form, i.e. no recrystallization of an active ingredient tookplace. In accordance with the prediction of the test system, a stablesolid solution of the active ingredients is present.

1. A liquid mixture comprising a) 1,3-bis(pyrrolidon-1-yl)butane and b) at least one compound of the formula I

 in which Q is CH₂-Z, CH₂—CH₂-Z or CHZ-C₁-C₄-alkyl, n is 0, 1, 2 or 3, and the Z radicals are C₁-C₂₀-alkylcarbonyloxy, carboxy, C₁-C₂₀-alkyloxycarbonyl, C₂-C₄-hydroxyalkyloxycarbonyl, di(C₁-C₄-alkyl)amino-C₂-C₄-alkyloxycarbonyl or tri(C₁-C₄-alkyl)ammonium-C₂-C₄-alkyloxycarbonyl.
 2. The mixture as claimed in claim 1, where component b) is 1,4-diacetoxybutane.
 3. The mixture as claimed in claim 1, in which components a) and b) are present in a ratio of from 10:1 to 1:10 by weight.
 4. A test system for evaluating the compatibility of a biologically active substance with a copolymer which includes units of N-vinyl-pyrrolidone and of ate least one ethylenic unsaturated monomer of the formula II CH₂═CR-Z′  (II) in which R is hydrogen or methyl, and Z′ has the meaning indicated in claim 1 for Z, where the test system includes a liquid mixture as claimed in claim 1 and at least one biologically active substance.
 5. The test system as claimed in claim 4, comprising from 10 to 70% by weight of biologically active substance.
 6. The test system as claimed in claim 4, additionally comprising at least one formulation aid.
 7. A method for evaluating the compatibility of a biologically active substance with an N-vinylpyrrolidone copolymer which includes units of N-vinylpyrrolidone in a proportion by weight of X_(VP) and units of at least one ethylenically unsaturated monomer of the formula II CH₂═CR-Z′  (II) in which R is hydrogen or methyl, Z′ is C₁-C₂₀-alkylcarbonyloxy, carboxyl, C₁-C₂₀-alkyloxycarbonyl, C₂-C₄-hydroxyalkyloxycarbonyl, di(C₁-C₄-alkyl)amino-C₂-C₄-alkyloxycarbonyl or tri(C₁-C₄-alkyl)ammonium-C₂-C₄-alkyloxycarbonyl, in a proportion by weight of X_(M), in which a) a test solvent which comprises 1,3-bis-(pyrrolidon-1-yl)butane in a proportion by weight of X_(VP) and a compound of the formula I in a proportion by weight of X_(M)

 in which Q is CH₂-Z, CH₂—CH₂-Z or CHZ-C₁-C₄-alkyl, n is 0, 1, 2 or 3, the Z radicals are identical and correspond to the Z′ radical, is prepared, b) the biologically active substance is brought into contact with the test solvent, and c) the phase behavior of the mixture and/or solubility of the biologically active substance in the test solvent is determined.
 8. The method as claimed in claim 7, in which the phase behavior of the mixture is investigated visually, spectroscopically and/or thermoanalytically.
 9. The method as claimed in claim 7, in which the mixture of biologically active substance and test solvent is heated to a temperature of up to 140° C. 